Project Summary Chronic stress has been implicated in depression, anxiety disorders, and various cognitive deficits. The underlying mechanisms are poorly understood but are thought to involve dendritic spine loss in limbic brain areas mediated in part by prolonged exposure to glucocorticoids. These hormones are released from the adrenal gland in response to stress, and they also oscillate in synchrony with the circadian rhythm. Recently, we have shown that glucocorticoids have rapid and potent effects on dendritic spine remodeling in superficial cortex. Still, very little is known about how stress affects this dynamic remodeling process in limbic areas or whether circadian glucocorticoid oscillations play any specific role in regulating it. How stress-induced changes in spines may relate to changes in functional connectivity in limbic circuitry is also unknown. The central hypothesis of this project is that circadian glucocorticoid oscillations regulate synaptic remodeling in limbic circuits by generating and stabilizing new spines, and that chronic stress leads to dysregulated remodeling and circuit dysfunction by disrupting these oscillations. Experiments to be conducted during the mentored phase will focus on the rapid effects of oscillating levels of glucocorticoid activity. Aim 1 will evaluate rapid effects on dendritic spine formation and elimination in infralimbic cortex using time-lapse microendoscopy, a recently developed tool for in vivo imaging of deep brain structures. Aim 2 will test for corresponding on functional connectivity, using optogenetic tools to stimulate amygdala projections to infralimbic cortex while measuring evoked activity using optrode recordings and functional magnetic resonance imaging. Research during the independent phase (Aim 3) will build on these results by evaluating the chronic effects of prolonged glucocorticoid exposure on spine remodeling, functional connectivity, and anxiety. In the process, the candidate will become proficient in the use of the tools described above, leveraging prior experience with similar methods. This will be accomplished under the supervision of a team of mentors and consultants (Deisseroth, Schatzberg. Raichle, de Lecea, Barretto) who pioneered these methods and have extensive experience training others to use them. They also have highly successful track records in preparing junior investigators for the transition to independence. We anticipate that this project will yield novel insights regarding the importance of circadian oscillations in glucocorticoid activity, particularly for stabilizing dendritic spines and preserving connectivity within limbic circuits. The results will also inform future efforts to study stress and glucocorticoid effects on functional connectivity in healthy human subjects and clinical populations.